Background & Motivation
Paper strip tests, where an analyte is measured by observing the change in colour of a dye in the test strip when it is dipped in the solution to be analysed have been highly developed and are very widely used. Some examples can make 10 simultaneous measurements covering a range of tests. The results are determined by comparing the developed colours with standard charts, which convert the colour to concentration values. This method is very useful but fits poorly with the digital age. The critical human intervention is a weak link that cannot survive current requirements for accuracy, repeatabiity & documentation.
The novel work presented here represents a step between the hi-tech lab approach & the low tech paper strip approach. Robust recording and documentation is integral to the method. Portability & low power are key requirements.
Here we present the design and construction of a simple, inexpensive apparatus to measure a wide range of enzyme reactions in a way that has been little used in the past (for reasons that will be explained later), by measuring conductivity changes (conductometric analysis).
We show that the design is simple to construct from cheap readily available materials.
We show that the apparatus is easy to use, works well and is reliable.
We suggest a range of uses & modifications that could be made
There is a need in developing countries for simple inexpensive lightweight diagnostic kit for blood & urine analysis that can be easily transported around by e.g. bicycle and operate with a low power requirement (e.g. photovoltaic) in a wide range of environments. Most diseases can be detected using biochemical tests, so they are very widely used in medicine in developed countries but not much in developing countries for reasons that will become apparent below.
THIS been the main way of MAKING CLINICAL TEST MEAUREMENTS but often requires that the analytical method has to be adapted to fit with the spectroscopic technique, making the measurement no longer the truly ‘Natural’ process. More later.
A good example of a test that has been developed for world-wide application is the test for blood glucose (i.e. diabetes) that in it’s simplest form comprises dipping a piece of paper in blood/ urine and looking for a colour change. The analysis of glucose is one of the most important and the present work makes no attempt to compete in this area as recent microelectronic microvolume test kits have revolutionised this area using conductivity- & charge based techniques. They are also inexpensive!
We demonstrate the measurement of urea in urine, a useful test for kidney function. The method has also been applied to measurement of alkaline phosphatase & AMP.
In developed countries it has become standard practice to base an expensive (£20k - £100+k) multipurpose analytical instrument at a geographical centre and send all samples from the region to this point. These machines are able to perform many different measurements (that relate to e.g. the status of blood, heart, muscle, lungs, excretion, infection, cancer) and to do so rapidly to get a quick turnaround on results. This provides a wide biochemical profile of the patient that is a remarkably effective indicator of health status.
This approach is often not appropriate, particularly in rural areas of developing countries, owing to transportation and access difficulties as well as financial considerations. It is better to travel cheaply to the patient and perform the analysis on site, together, if possible, with a diagnosis from which it may be possible to decide what treatment a patient needs, i.e. do they need to go to hospital? This latter may be difficult for them.
Most clinical analyses use enzyme catalysts to measure the concentration of biochemicals. Antibody analyses are also very important but are not covered here.
Enzymes, which are proteins, accelerate the rate of every chemical reaction in humans and in every other living organism. The reactions (e.g. the hydrolysis of ATP to allow muscle contraction) would not occur at 37 deg. C without catalysis & so enzymes are vital for every aspects of life. Some tests use antibodies to measure levels of proteins.
Measurements of the concentrations (& changes therein) of biochemical compounds (e.g. ATP) by using changes in solution conductivity (see below), have been little used because it is necessary to control the temperature of the reaction cell very accurately, to within approx. +/- 0.010 deg C. This has generally been perceived to be very difficult to achieve, particularly in a simple cheap apparatus. A stability of +/- 0.02 deg C has been achieved, for the time required for a series of reaction measurements, in a simple and inexpensive manner – thus the main hurdle that must be addressed for the use of this technology has been surmounted in a simple way.
Conductivity, the flow of an electric current through a solution, can be used to measure most biochemical reactions since they nearly all involve a change in the ionic charge when the reaction takes place.
This is explained briefly below but here we will take a brief look at conductivity itself.
An electric current in a copper wire is carried by negatively charged electrons, which jump from one copper atom to another as they flow through the wire. They travel from the negative terminal (neutral) to the positive (live) terminal. In a liquid solution the electrons are carried through the solution as ions (much more slowly as they have to diffuse through the liquid, under the influence of the electric field).
The current passes from the cathode (negative) to the anode (positive). The current flow at a fixed voltage is dependent on the number of ions present, as normally an ion can carry only one electron. In this work all measurents are made by determining the magnitude of the current that flows between the cathode and anode, i.e. this gives a measurement of the number of ions present in the test solution.
Ions form in solution as salts dissolve in water. Thus sodium chloride, common salt, NaCl, in which the sodium & chlorine atoms are uncharged in the solid crystalline state, completely forms Na+ and Cl- ions when dissolved in water. It is these ions which conduct (carry) the electric current in solution. The crystals are an insulator because there are no charges that can move in the crystal & they thus do not conduct electricity. Note that the positive ions move in the opposite direction to the negative ions.
The ions Na+ and Cl- form when sodium chloride dissolves because these interact strongly with water and are stabilised relative to the uncharged salt.
Enzymes speed up chemical reactions & catalyse all reactions in all living organisms. Thus they are obviously essential for life. They are extremely efficient & are composed of large protein molecules that are quite delicate outside the organism. An example of an enzyme reaction (urease) is given later. Good examples of their action is in digestion where large complex food molecules (e.g. starch) are reduced to small molecules that can be absorbed from the gut into the bloodstream & so used to generate energy in the body for muscle contraction, nerve conduction etc.
Enzymes work by recognising and binding the substrate – S the reactant molecule. The complex that forms [ES] then converts into an enzyme-product complex.
This then dissociates to give the enzyme back (it is a catalyst, so it has to be recovered to act many times) and the product - P of the reaction.
This can be represented symbolically as:
E + S <----> [ES] -----> E + P
Binding Km Catalysis kcat
For details click link to Enzymes
The enzyme catalysis is analysed by measuring the rate of the reaction at several different values of the substrate concentration [S]. These values are then analysed using a graphical plot to determine the strength of the binding Km and the efficiency of the catalysis part kcat. This interpretation of enzyme action is known as Michaelis-Menten kinetic analysis & is very widely used in biochemistry generally and needs to be done thoroughly to design a clinical test, such as that for urea in urine or blood.
Enzyme reaction rates are usually measured at a series of substrate concentrations, as initial velocities (vi) i.e. only the first part of the reaction is measured – before the amount of substrate has changed significantly. This gives a straight line for product formation that is easy to interpret. The plot that is often used is the Lineweaver-Burke plot of 1/vi against 1/[S] but a plot of [S]/vi against [S] is better & will be seen later.
The enzyme reaction rate is described (in terms of initial velocities vi) by the Michaelis-Menten equation (derived from the model shown above):
vi = kcat . [Eo] . [S]/(Km + [S])
where [S] is the concentration of substrate, Km describes the binding of the substrate to the enzyme, kcat is the rate at which the product is formed from the bound substrate [ES] & [Eo] is the enzyme concentration. The equation describes a saturation curve that implies that when all the enzyme has bound substrate (at high [S]), the enzyme reaction cannot go any faster – this condition is referred to as the Maximum Velocity.
An example of this analysis, using an [S]/vi vs. [S] plot (which gives a straight line) for the urease reaction, is given later in the RESULTS section.